This invention relates to a TRAF family molecule, a polynucleotide encoding the molecule, an antibody against the molecule, and an antisense polynucleotide of the polynucleotide encoding the molecule.
For molecules belonging to the TNF receptor family (hereinafter referred to as xe2x80x9cTNF-Rxe2x80x9d), TNF-R1, TNF-R2, Fas, CD40, CD27, and lymphotoxin-xcex2 receptors (hereinafter referred to as xe2x80x9cLT-xcex2Rxe2x80x9d or xe2x80x9cTNF-Rrpxe2x80x9d) are known. These molecules are provided with the function of finally causing the phenomena of the proliferation, cell differentiation, and necrosis of cells by binding to their ligands, or with the function as second signal molecules of other molecules.
Also, TRAF1 and TRAF2 (the abbreviation of TNF-R associated factor), which are molecules capable of associating TNF-R, have been cloned (Goeddel et. al, Cell Vol. 78, No. 4, p. 681-692, 1994) and these are known to associate the domain within the cytoplasm of TNF-R2.
Furthermore, two additional kinds of new TRAF molecules have been identified and the following are noted for these molecules:
(1) TRAF1 associates TNF-R2 and CD40. (J. Biol. Chem. Vol. 269, No. 48, p. 30069-30072.)
(2) TRAF2 plays an important role in activation of the DNA-binding of NF-xcexaB by TNF-R2 or CD40: NF-xcexaB is a nucleoprotein binding to a base sequence comprising 10 base pairs that is within the enhancer region of the Igxcexa L chain gene and is referred to as xe2x80x9cxcexaB motifxe2x80x9d.
(3) TRAF3 (also referred to as xe2x80x9cCRAF1,xe2x80x9d xe2x80x9cCD40bp,xe2x80x9d or xe2x80x9cCAP1xe2x80x9d) is known to associate TNF-R2, CD40, and LT-xcex2R. (Science Vol. 267, No. 5203, p. 1494-1498; FEBS Lett. Vol. 358, No. 2, p. 113-118; J. Biol. Chem. Vol. 269, p.30069-30072; and Cell Vol. 80, No. 3, P. 389-399.)
(4) As for TRAF4 (also referred to as xe2x80x9cCART1xe2x80x9d), no molecule has yet been identified to associate it. (J. Biol. Chem. Vol. 270, No. 4, P. 25715-25721, 1995.)
Moreover, it has been reported that neither TRAF1 nor TRAF3 induces activation of the DNA-binding of NF-xcexaB. (Cell Vol. 81, p.495-504; Science Vol. 269, p.1424-1427; and Proc. Natl. Acad. Sci. USA, Vol. 93, p.9699-9703.)
Molecules of the TRAF family are structurally characterized by having TRAF domains, and some members of the family molecules have ring finger motifs, Zn finger motifs or leucine zipper motifs at their N-termini, or coiled coil structures at their centers. Because of these structural features, it is presumed that these TRAF family molecules form complexes in a homo- or hetero-fashion and function as transcription factors.
An object of this invention is to provide a novel TRAF family molecule. Another object of the invention is to provide a substance capable of having an application in the elucidation of functions of the TRAF family molecules, such as an antibody or a polynucleotide probe: namely, the elucidation of interaction between the TRAF family molecules and the elucidation of the signal transduction system of the TNF-R family. A further object of the invention is to provide an antisense polynucleotide applicable to the development of pharmaceuticals.
Specifically, an object of the invention is to provide the screening for the TRAF5 molecule, which is a novel TRAF family molecule according to the invention, isolation and structural determination thereof, as well as to provide a variety of functions that the molecule possesses.
In other words, the object of the invention is as follows: homology with respect to TRAF1 and TRAF2 is analyzed by computer; various oligo-DNA primers capable of amplifying the region that is most highly conserved are synthesized; cDNA is prepared from RNA that is derived from different kinds of cell strains and tissues; PCR is performed using the oligo-DNA primers and the cDNA as a template; and the thus obtained novel gene (TRAF5 gene) exhibiting a high degree of homology to the TRAF1 and TRAF2 genes as well as the amino acid sequence of the TRAF5 molecule encoded by the gene is provided.
In the present specification, the protein will be hereinafter referred to as xe2x80x9cTRAF5 moleculexe2x80x9d or xe2x80x9cTRAF5xe2x80x9d and the TRAF5 gene itself referred to as xe2x80x9cTRAF5 gene.xe2x80x9d
It is also an object of the invention to provide information that the TRAF5 molecule tends to associate LT-xcex2R and CD30 and further that it has the ability to induce the DNA-binding activity of NF-xcexaB, which is a DNA-binding protein.
In addition, an object of the invention is to provide means for elucidating the functions of the TRAF5 molecule as well as to provide means for elucidating the signal transduction system of the TNF-R family involving the TRAF family molecules by preparing antibodies against the TRAF5 molecule and using the antibodies.
A still further object of the invention is to provide means for making the TRAF5 gene applicable to experimental probes, probes diagnosing the gene or the antisense genes, and therapeutic agents.
More specifically, this invention provides novel TRAF family molecules as described below, including the TRAF5 molecule.
1. A TRAF family molecule having the properties as noted in (1)-(4) in the following:
(1) Having the ability to associate lymphotoxin-xcex2receptors (LT-xcex2R) and CD30;
(2) Having the ability to induce the DNA-binding activity of a DNA-binding protein;
(3) Having the inability to associate CD40 or TNF-R2; and
(4) Having a leucine zipper motif or coiled coil structure within its molecule.
2. The TRAF family molecule as described above wherein the DNA-binding protein is NF-xcexaB.
3. The TRAF family molecule as described above wherein said molecule is a protein encoded by DNA hybridizing to the base sequence set forth in SEQ ID No. 2 of the Sequence Listing under stringent conditions.
4. The TRAF family molecule as described above wherein said molecule has at least part of the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 3 of the Sequence Listing.
5. A TRAF5 molecule comprising the amino acid sequence set forth in SEQ ID No. 1 of the Sequence Listing.
6. A TRAF5 molecule comprising the amino acid sequence set forth in SEQ ID No. 3 of the Sequence Listing.
7. A TRAF family molecule comprising a modified TRAF5 molecule, said TRAF5 molecule comprising the amino acid sequence which is derived by modifying a part of the amino acid sequence set forth in SEQ ID No. 1 or SEQ ID No. 3 of the Sequence Listing, by substitution, deletion or addition of amino acid(s) without substantially altering the functions of the TRAF family molecule.
8. A TRAF5 molecule comprising an amino acid sequence of from position 233 to position 558 of the amino acid sequence set forth in SEQ ID No. 1 of the Sequence Listing.
9. A TRAF5 molecule comprising an amino acid sequence of from position 342 to position 558 of the amino acid sequence set forth in SEQ ID No. 1 of the Sequence Listing.
10. A TRAF5 molecule comprising an amino acid sequence of from position 233 to position 557 of the amino acid sequence set forth in SEQ ID No. 3 of the Sequence Listing.
11. A TRAF5 molecule comprising an amino acid sequence of from position 342 to position 557 of the amino acid sequence set forth in SEQ ID No. 3 of the Sequence Listing.
12. A polynucleotide encoding the TRAF family molecule as described above.
13. A TRAF5 gene comprising the base sequence set forth in SEQ ID No. 2 of the Sequence Listing.
14. A TRAF5 gene comprising the base sequence set forth in SEQ ID No. 4 of the Sequence Listing.
15. A polynucleotide comprising part of the polynucleotide according to any of Items 12-14 as described above, said part being 12 or more consecutive bases.
16. A polynucleotide comprising part of the polynucleotide according to any of Items 12-14 as described above, said part being 16 or more consecutive bases.
17. An antibody directed against the TRAF family molecule according to any of Items 1-11 as described above.
18. An expression vector comprising a polynucleotide encoding the TRAF family molecule according to Item 12 as described above.
19. A transformant characterized by being transformed with the expression vector as described above.
20. A method of producing a TRAF family molecule, said method comprising using the transformant as described above.
21. An antisense polynucleotide of a polynucleotide encoding the TRAF family molecule according to Item 12 as described above.
22. A polynucleotide comprising part of the antisense polynucleotide as described above, said part being 12 or more consecutive bases.
23. A polynucleotide comprising part of the antisense polynucleotide as described above, said part being from 16 or more to 30 or less consecutive bases.
24. A derivative of the polynucleotide according to any of Items 12-16 or Items 21-23 as described above.